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Somatostatin receptor subtype 5 (SST5 ) is an emerging biomarker and actionable target in pituitary (PitNETs) and pancreatic (PanNETs) neuroendocrine tumors. Transcriptional and epigenetic regulation of SSTR5 gene expression and mRNA biogenesis is poorly understood. Recently, an overlapping natural antisense transcript, SSTR5-AS1, potentially regulating SSTR5 expression, was identified. We aimed to elucidate whether epigenetic processes contribute to the regulation of SSTR5 expression in PitNETs (somatotropinomas) and PanNETs. We analyzed the SSTR5/SSTR5-AS1 human locus in silico to identify CpG islands. SSTR5 and SSTR5-AS1 expression was assessed by quantitative real-time PCR (qPCR) in 27 somatotropinomas, 11 normal pituitaries (NPs), and 15 PanNETs/paired adjacent (control) samples. We evaluated methylation grade in four CpG islands in the SSTR5/SSTR5-AS1 genes. Results revealed that SSTR5 and SSTR5-AS1 were directly correlated in NP, somatotropinoma, and PanNET samples. Interestingly, selected CpG islands were differentially methylated in somatotropinomas compared with NPs. In PanNETs cell lines, SSTR5-AS1 silencing downregulated SSTR5 expression, altered aggressiveness features, and influenced pasireotide response. These results provide evidence that SSTR5 expression in PitNETs and PanNETs can be epigenetically regulated by the SSTR5-AS1 antisense transcript and, indirectly, by DNA methylation, which may thereby impact tumor behavior and treatment response., This research was funded by Junta de Andalucía (BIO-0139, P20_00442; PEER-0048-2020); Spanish Ministry of Economy (BFU2016-80360-R), Ministry of Science and Innovation (PID2019-105201RB-I00, PID2019-105564RB-I00); and ISCIII (PI16-00264, CD19/00255), co-funded with EU funds from FEDER Program. People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under project WHRI-ACADEMY, REA grant agreement n° 608765; MECD (FPU14/04290, FPU18/02275); EMBO (short term fellowship 6802); GETNE G2019 Research Grant; Fundación Eugenio Rodriguez Pascual (FERP2019); project grants from the UK Medical Research Council (MR/R022836/1; MR/L002345/1); and CIBERobn; CIBER Fisiopatología de la Obesidad y Nutriciín is an initiative of Instituto de Salud Carlos III.

10 pags., 4 figs., 5 tabs., Objective: The aim of this study was to quantify the changes obtained when the molar intrusion movement is complemented by photobiomodulation (PBM). Background: A common problem in adult patients is the super-eruption of maxillary molars caused by the loss of the antagonist tooth. Super-erupted molars impair oral rehabilitation and can cause both occlusal and functional problems. There is increasing research confirming the benefits of adjunctive PBM during orthodontic treatment. Methods: Twenty patients with indication of a maxillary first molar intrusion for oral rehabilitation were selected. Patients were randomized into two groups to receive orthodontic intrusion (control group) or the same treatment complemented by PBM (PBM group) in repeated doses (days 0, 1, 2, 3, 4, and 7 from the start of the intrusion and in each monthly follow-up) by using a low-power red laser diode (670 nm, 150 mW, 12 min around the molar). Plaque index (PI), probing depth (PD), and bleeding of probing (BOP) were assessed at 0, 1, 2, 3, and 6 months. Stereolithography models generated from an intraoral scanner were taken at 0, 3, and 6 months and cone beam computed tomography (CBCT) records were taken at 0 and 6 months. Mean intrusion distance, mean intrusion velocity, and volumetric resorption were calculated. Results: Periodontal clinical assessments (PI, PD, and BOP) and mean intrusion distance or mean intrusion velocity yielded no differences ( p > 0.05) between groups. However, PBM group showed lower values of all these scores during the first 3 months. Intraoral scanner and CBCT were equally effective in accurately monitoring the intrusion distance ( p > 0.05). CBCT records allowed volumetric evaluation of the root resorption process, being lesser in the PBM group, but not significantly ( p > 0.05). Conclusions: During orthodontic intrusion process, the adjunctive application of PBM may provide better periodontal records and lower progression of root resorption at the expense of a little lower intrusion distance and velocity., This research was supported by a grant from the Eugenio Rodrıguez Pascual Foundation (Madrid, Spain) and by the Spanish Research Projects of MICINN (Ref. MAT 2017- 83856-C3 and Ref. PiD2020-114755GB-C31).

[Rationale] SPINOPHILIN (SPN, PPP1R9B) is an important tumor suppressor involved in the progression and malignancy of different tumors depending on its association with protein phosphatase 1 (PP1) and the ability of the PP1-SPN holoenzyme to dephosphorylate retinoblastoma (pRB)., [Methods] We performed a mutational analysis of SPN in human tumors, focusing on the region of interaction with PP1 and pRB. We explored the effect of the SPN-A566V mutation in an immortalized non-tumorigenic cell line of epithelial breast tissue, MCF10A, and in two different p53-mutated breast cancer cells lines, T47D and MDA-MB-468., [Results] We characterized an oncogenic mutation of SPN found in human tumor samples, SPN-A566V, that affects both the SPN-PP1 interaction and its phosphatase activity. The SPN-A566V mutation does not affect the interaction of the PP1-SPN holoenzyme with pocket proteins pRB, p107 and p130, but it affects its ability to dephosphorylate them during G0/G1 and G1, indicating that the PP1-SPN holoenzyme regulates cell cycle progression. SPN-A566V also promoted stemness, establishing a connection between the cell cycle and stem cell biology via pocket proteins and PP1-SPN regulation. However, only cells with both SPN-A566V and mutant p53 have increased tumorigenic and stemness properties., [Conclusions] SPN-A566V, or other equivalent mutations, could be late events that promote tumor progression by increasing the CSC pool and, eventually, the malignant behavior of the tumor., This work was supported by grants from the Ministerio de Ciencia, Innovación y Universidades (MCIU) Plan Estatal de I+D+I 2018, a la Agencia Estatal de Investigación (AEI) y al Fondo Europeo de Desarrollo Regional (MCIU/AEI/FEDER, UE): RTI2018-097455-B-I00 and RTI2018-096735-B-I00 grant from AEI-MICIU/FEDER (RED2018-102723-T); from CIBER de Cáncer (CB16/12/00275), co-funded by FEDER from Regional Development European Funds (European Union); from Consejeria de Salud (PI-0397-2017) and Consejeria of Economía, Conocimiento, Empresas y Universidad of the Junta de Andalucia (P18-RT-2501). Special thanks are also due to the Fundacion AECC and Fundacion Eugenio Rodriguez Pascual for supporting this work.

10 pags., 4 figs., 2 tabs., Background Photodynamic therapy (PDT) kills target microorganisms via reactive oxygen species (ROS) production. PDT seems to be a good alternative treatment option for onychomycosis. Objective To compare the efficacy of combined therapies based on oral terbinafine (TN) plus adjunctive PDT mediated by methylene blue (MB) (TN + MB/PDT) or methyl aminolevulinate (MAL) (TN + MAL/PDT) in the treatment of onychomycosis. Methods Twenty patients affected by severe dermatophyte onychomycosis in the nails of the big toe (>60% disease involvement of target nail) received oral TN for 12 weeks and concomitantly were randomly allocated to receive nine sessions, separated by 2‐week intervals, of urea (40%) plus a PDT protocol mediated by MB (TN + MB/PDT: group I) or mediated by MAL (TN + MAL/PDT: group II). Clinical and mycological efficacy was evaluated at 16‐, 40‐ and 52‐week follow‐up., Results Both protocols showed a significant decrease in Onychomycosis Severity Index (OSI) scores (P < .05), from 24.2 ± 4.6 to 0.7 ± 0.6 (group I)) and from 18.5 ± 10.1 to 2.1 ± 2.0 (group II). No side effects or complications were reported in any of the combinations used. Mycological cure rates were significantly higher during the last third of the evaluated period of time, reaching 100% and 90% in group I and group II, respectively, at the 52‐week follow‐up. In both modalities, complete cure was achieved in 70% of the patients at the 52‐week follow‐up. Conclusions TN + MB/PDT and TN + MAL/PDT show similar outcomes in the treatment of toenails with severe onychomycosis. PDT is an effective method to accelerate the TN‐mediated healing process., This research was supported by a grant from the Eugenio Rodríguez Pascual Foundation (Madrid, Spain) and by the Spanish Research Project MICINN (Ref.: MAT 2017‐83856‐C3).

5 pags., 3 figs., Background: Although systemic therapies are recommended for severe or recalcitrant cases of pityriasis versicolor (PV), they are not free of important side effects and drug interactions. Photodynamic therapy (PDT) utilizes the action of singlet oxygen and free radicals produced by a light-activated photosensitizer to kill viruses, bacteria, or fungi. In this study, the effect of a PDT mediated by methylene blue (MB) in PV was evaluated. Methods: Five women with PV disseminated on the back and diagnosed by fresh microscopic analysis were treated with a solution of MB (2%) applied to the PV lesions for 3 minutes. Next, a red LED lamp (λ = 630±5 nm, 37 J/cm), placed 100 mm from the skin for 10 minutes, was applied on the dyed PV lesions. Six sessions of MB/PDT were implemented with a 2-week interval in between. Wood's lamp examination was used to monitor fungal infection at each time point. Results: Complete cure was observed in the five women at the 4 weeks post-treatment follow-up. Fluoresce images from PV lesions by Wood's lamp allowed to evaluate whether the lesions were healed or not at each time point. No patient showed relapse at the 6-month follow-up. The patients did not have any adverse effect, and good cosmetic outcome was observed. Conclusions: Six sessions of MB/PDT spaced at 14-day intervals are sufficient for the treatment for PV in healthy patients., Spanish Research Projects MINECO. Grant Number: MAT 2017‐83856‐C3 Fundación Eugenio Rodríguez Pascual

Cellular senescence is a stress-response mechanism that contributes to homeostasis maintenance, playing a beneficial role during embryogenesis and in normal adult organisms. In contrast, chronic senescence activation may be responsible for other events such as age-related disorders, HIV and cancer development. Cellular senescence activation can be triggered by different insults. Regardless of the inducer, there are several phenotypes generally shared among senescent cells: cell division arrest, an aberrant shape, increased size, high granularity because of increased numbers of lysosomes and vacuoles, apoptosis resistance, defective metabolism and some chromatin alterations. Senescent cells constitute an important area for research due to their contributions to the pathogenesis of different diseases such as frailty, sarcopenia and aging-related diseases, including cancer and HIV infection, which show an accelerated aging. Hence, a new pharmacological category of treatments called senotherapeutics is under development. This group includes senolytic drugs that selectively attack senescent cells and senostatic drugs that suppress SASP factor delivery, inhibiting senescent cell development. These new drugs can have positive therapeutic effects on aging-related disorders and act in cancer as antitumor drugs, avoiding the undesired effects of senescent cells such as those from SASP. Here, we review senotherapeutics and how they might affect cancer and HIV disease, two very different aging-related diseases, and review some compounds acting as senolytics in clinical trials., This work was supported by grants from the Ministerio de Ciencia, Innovación y Universidades (MCIU) Plan Estatal de I+D+I 2018, a la Agencia Estatal de Investigación (AEI) y al Fondo Europeo de Desarrollo Regional (MCIU/AEI/FEDER, UE): RTI2018-097455-B-I00; grant from AEI-MICIU/FEDER (RED2018-102723-T); from CIBER de Cáncer (CB16/12/00275), co-funded by FEDER from Regional Development European Funds (European Union); from Consejeria de Salud (PI-0397-2017) andProject P18-RT-2501 from 2018 competitive research projects call within the scope of PAIDI 2020—80% co-financed by the European Regional Development Fund (ERDF) from the Regional Ministry of Economic Transformation, Industry, Knowledge and Universities. Adalucia move with Europe. Additionally, we thank the Fundacion AECC and Fundacion Eugenio Rodriguez Pascual for supporting this work. LS-D and ES-M were funded by grants from Ministerio de Ciencia, Innovación y Universidades (MCIU) Plan Estatal de I+D+I 2018, a la Agencia Estatal de Investigación (AEI) y al Fondo Europeo de Desarrollo Regional (MCIU/AEI/FEDER, UE). AE-S was funded by Fundacion AECC.

Endothelial adenosine monophosphate-activated protein kinase (AMPK) plays a critical role in the regulation of vascular tone through stimulating nitric oxide (NO) release in endothelial cells. Since obesity leads to endothelial dysfunction and AMPK dysregulation, AMPK activation might be an important strategy to restore vascular function in cardiometabolic alterations. Here, we report the identification of a novel AMPK modulator, the indolic derivative IND6, which shows affinity for AMPKα1β1γ1, the primary AMPK isoform in human EA.Hy926 endothelial cells. IND6 shows inhibitory action of the enzymatic activity in vitro, but increases the levels of p-Thr174AMPK, p-Ser1177eNOS and p-Ser79ACC in EA.Hy926. This paradoxical finding might be explained by the ability of IND6 to act as a mixed-type inhibitor, but also to promote the enzyme activation by adopting two distinct binding modes at the ADaM site. Moreover, functional assays reveal that IND6 increased the eNOS-dependent production of NO and elicited a concentration-dependent vasodilation of endothelium-intact rat aorta due to AMPK and eNOS activation, demonstrating a functional activation of the AMPK–eNOS–NO endothelial pathway. This kinase inhibition profile, combined with the paradoxical AMPK activation in cells and arteries, suggests that these new chemical entities may constitute a valuable starting point for the development of new AMPK modulators with therapeutic potential for the treatment of vascular complications associated with obesity., his research was funded by Fundación Eugenio Rodríguez Pascual, the Spanish Ministerio de Economía y Competitividad (MDM-2017-0767), the Spanish Ministerio de Ciencia Innovación y Universidades (RTI2018- 095544-B-I00) and the Generalitat de Catalunya (2017SGR1746) for financial support. Computational resources from the Barcelona Supercomputing Center (BSC; BCV-2019-1-0009 and BCV-2019-2-0017) and the Consorci de Serveis Universitaris de Catalunya (CSUC; Molecular Recognition project) are acknowledged. E.A. thanks AGAUR (Generalitat of Catalunya; 2019FI_B2_00001) for her fellowship. M.S.G. thanks Universidad Com- plutense de Madrid for her fellowship.

Background and Aims: Hepatocellular dedifferentiation is emerging as an important determinant in liver disease progression. Preservation of mature hepatocyte identity relies on a set of key genes, predominantly the transcription factor hepatocyte nuclear factor 4α (HNF4α) but also splicing factors like SLU7. How these factors interact and become dysregulated and the impact of their impairment in driving liver disease are not fully understood. Approach and Results: Expression of SLU7 and that of the adult and oncofetal isoforms of HNF4α, driven by its promoter 1 (P1) and P2, respectively, was studied in diseased human and mouse livers. Hepatic function and damage response were analyzed in wild-type and Slu7-haploinsufficient/heterozygous (Slu7) mice undergoing chronic (CCl) and acute (acetaminophen) injury. SLU7 expression was restored in CCl-injured mice using SLU7-expressing adeno-associated viruses (AAV-SLU7). The hepatocellular SLU7 interactome was characterized by mass spectrometry. Reduced SLU7 expression in human and mouse diseased livers correlated with a switch in HNF4α P1 to P2 usage. This response was reproduced in Slu7 mice, which displayed increased sensitivity to chronic and acute liver injury, enhanced oxidative stress, and marked impairment of hepatic functions. AAV-SLU7 infection prevented liver injury and hepatocellular dedifferentiation. Mechanistically we demonstrate a unique role for SLU7 in the preservation of HNF4α1 protein stability through its capacity to protect the liver against oxidative stress. SLU7 is herein identified as a key component of the stress granule proteome, an essential part of the cell’s antioxidant machinery. Conclusions: Our results place SLU7 at the highest level of hepatocellular identity control, identifying SLU7 as a link between stress-protective mechanisms and liver differentiation. These findings emphasize the importance of the preservation of hepatic functions in the protection from liver injury., Supported by MINECO/AEI/FEDER (UE SAF2016-75972-R, PID2019-104265RB-I00/AEI/10.13039/501100011033, and PID2019-104878RB-100/AEI/10.13039/501100011033), CIBERehd, Fundación La Caixa (HEPACARE), an AECC postdoctoral fellowship (POSTD18014AREC, to M.A.), a Ministerio de Educación FPU fellowship (FPU18/01461, to M.G.R.), a Ministerio de Educación FPI fellowship (BES-2017-079883, to M.R.); a Ramón y Cajal Program contract (RYC2018-024475-1, to M.G.F.B.), the Fundación Eugenio Rodríguez Pascual, the Fundación Mario Losantos, the Fundación M. Torres, and a generous donation from Mr. Eduardo Avila.

5 pags., 3 figs., 2 tabs., Background: Although surgical removal is the treatment of choice in Bowen's disease (BD), there are cases in which by age, comorbidities, use of anticoagulants, location, cosmetic result, or size, it is preferable to use other treatments such as cryotherapy, 5-fluorouracil cream, imiquimod 5% cream or photodynamic therapy (PDT). Efficacy of PDT in BD is supported by substantial research and clinical data. Objectives: This study aimed to evaluate the long term effectiveness of methyl aminolevulinate-PDT (MAL/PDT)on a wide range of Bowen lesions in different locations and sizes. Methods: Patients diagnosed with BD were treated in 3 sessions with a 4-week interval in between with MAL/PDT between January 2016 and January 2017 in a private clinic. Clinical response and relevant patient and tumour characteristics were analyzed during the first year after start of the PDT sessions. Results: In total, 21 BD lesions in 18 patients were included in the study. Complete regression (CR)after 3rd PDT session was 87.5% and 100% at the 6-month follow-up. Treatment was well tolerated and local adverse reactions were very scarce. No recurrence was observed at 12-month follow-up. Cosmetic outcome at 12 months was good or excellent in 100% of patients. Conclusions: MAL/PDT is an effective, non invasive and safe treatment modality for BD with excellent cosmesis., This research was supported by a grant from the Eugenio Rodríguez Pascual Foundation (Madrid, Spain) and by the Spanish ResearchProject MINECO (Ref.: MAT 2017-83856-C3).

6 pags., 3 figs., 3 tabs., Background: The increase in the number of basal cell carcinoma (BCC) lesions has prompted use of minimally invasive therapies, including Photodynamic therapy (PDT). . The objective of the present work was to analyze the efficacy of methyl aminolevulinate-mediated photodynamic therapy (MAL-PDT) in patients suffering from superficial or nodular BCCs. Methods: A total of 220 BCC lesions (76 superficial and 144 nodular), clinically diagnosed and confirmed by histopathology analysis, were treated in 174 patients (mean age 72.5). Debulking using curettage was performed before two or three MAL-PDT sessions (λ = 630 nm; 90 J/cm; 23 min) at 4-week intervals. Analyses of clinical clearance and cosmetic outcome were carried out by direct examination, dermoscopy, photographs, as well as by fluorescence diagnosis using a Wood's lamp. Evaluations were carried out at the different PDT sessions and follow-ups over a 3-year period. Results: MAL-PDT was safe and highly tolerated. After an average of 2.6 MAL-PDT sessions, the overall clearance rate at 3-year follow-up was 96.1 % (95 % confidence interval [CI] 100 %–92 %) for superficial BCCs and 95.2 % (95 % [CI] 99 %-92 %) for nodular BCCs after an average of 2.7 sessions. Minimal side effects such as crushing, erythema and edema were reported. All BCC lesions showed excellent or good cosmetic results. Conclusion: The protocol followed in the present study has shown that MAL-PDT is a safe and effective treatment for superficial and nodular types of BCC., This research was supported by a grant from the Eugenio Rodríguez Pascual Foundation (Madrid, Spain) and by the Spanish Research Project MINECO (Ref.: MAT 2017-83856-C3).

Sarcomas constitute a rare heterogeneous group of tumors, including a wide variety of histological subtypes. Despite advances in our understanding of the pathophysiology of the disease, first-line sarcoma treatment options are still limited and new treatment approaches are needed. Histone H2AX phosphorylation is a sensitive marker for double strand breaks and has recently emerged as biomarker of DNA damage for new drug development. In this study, we explored the role of H2AX phosphorylation at Ser139 alone or in combination with MAP17 protein, an inducer of DNA damage through ROS increase, as prognostic biomarkers in sarcoma tumors. Next, we proposed doxorubicin and olaparib combination as potential therapeutic strategies against sarcomas displaying high level of both markers. We evaluate retrospectively the levels of pH2AX (Ser139) and MAP17 in a cohort of 69 patients with different sarcoma types and its relationship with clinical and pathological features. We found that the levels of pH2AX and MAP17 were related to clinical features and poor survival. Next, we pursued PARP1 inhibition with olaparib to potentiate the antitumor effect of DNA damaging effect of the DNA damaging agent doxorubicin to achieve an optimal synergy in sarcoma. We demonstrated that the combination of olaparib and doxorubicin was synergistic in vitro, inhibiting cell proliferation and enhancing pH2AX intranuclear accumulation, as a result of DNA damage. The synergism was corroborated in patient-derived xenografts (PDX) where the combination was effective in tumors with high levels of pH2AX and MAP17, suggesting that both biomarkers might potentially identify patients who better benefit from this combined therapy., The AC lab was supported by grants from the Ministerio de Ciencia, Innovación y Universidades (MCIU) Plan Estatal de I + D + I 2018, Agencia Estatal de Investigación (AEI) and (Regional Development European Funds (FEDER): RTI2018–097455-B-I00 (MCIU/AEI/FEDER, UE); and CIBER de Cáncer (CB16/12/00275), co-funded by FEDER from Regional Development European Funds (European Union). SMG was funded by Consejería de Salud of the Junta de Andalucía (PI-0397-2017) and the Fundación AECC. Especial thanks to the Fundación AECC and Fundación Eugenio Rodriguez Pascual for supporting this work. The authors thank the HUVR-IBiS Biobank (Andalusian Public Health System Biobank and ISCIII-Red de Biobancos PT17/0015/0041) for the assessment and technical support provided.

8 pags., 3 figs. 3 tabs., Photodynamic therapy (PDT) has emerged as an interesting alternative option for onychomycosis treatment. The impact of a specific photosensitizer (PS) on the final result is an important factor to consider. We conducted a short- and medium-term controlled trial to compare the effectiveness of PDT in the treatment of mild-to-moderate onychomycosis when it is mediated by two different PSs. Twenty patients were randomized to receive nine sessions of PDT distributed over 16 weeks mediated either by methylene blue (MB/PDT group) or methyl aminolevulinate (MAL/PDT group). Onychomycosis severity index (OSI) and nail involvement were checked along the study. Complete cure, treatment success, and clinical improvement were tabulated at 16 and 40-week follow-ups. OSI scores decreased significantly along the study, from 12.1 ± 5.4 to 3.6 ± 3.2 (MB/PDT group) and from 14.8 ± 6.0 to 5.4 ± 4.4 (MAL/PDT group). At 16-week follow-up, only 20% of the patients in the MB/PDT group reached complete cure and none in the group of MAL/PDT. At 40-week follow-up, complete cure rates were 70% and 40% in the MB/PDT group and MAL/PDT group respectively. Both modalities showed good outcomes in treatment of moderate toenail onychomycosis. MB/PDT showed a faster action but with relapse rates slightly higher than MAL/PDT., This research was supported by a grant from the Eugenio Rodríguez Pascual Foundation (Madrid, Spain) and by the Spanish Research Project MICINN (Ref.: MAT 2017-83856-C3)

This article belongs to the Special Issue Cancer Stem Cells and Resistance to Therapy., Cancer stem cells (CSC) are associated with the mechanisms of chemoresistance to different cytotoxic drugs or radiotherapy, as well as with tumor relapse and a poor prognosis. Various studies have shown that mitochondria play a central role in these processes because of the ability of this organelle to modify cell metabolism, allowing survival and avoiding apoptosis clearance of cancer cells. Thus, the whole mitochondrial cycle, from its biogenesis to its death, either by mitophagy or by apoptosis, can be targeted by different drugs to reduce mitochondrial fitness, allowing for a restored or increased sensitivity to chemotherapeutic drugs. Once mitochondrial misbalance is induced by a specific drug in any of the processes of mitochondrial metabolism, two elements are commonly boosted: an increment in reactive nitrogen/oxygen species and, subsequently, activation of the intrinsic apoptotic pathway., This research was funded by grants from the Ministerio de Ciencia, Innovación y Universidades (MCIU) Plan Estatal de I+D+I 2018, a la Agencia Estatal de Investigación (AEI) y al Fondo Europeo de Desarrollo Regional (MCIU/AEI/FEDER, UE): RTI2018-097455-B-I00; grant from AEI-MICIU/FEDER (RED2018-102723-T); from CIBER de Cáncer (CB16/12/00275), co-funded by FEDER from Regional Development European Funds (European Union); from Consejería de Salud (PI-0397-2017) and Consejería of Economía, Conocimiento, Empresas y Universidad of the Junta de Andalucía (P18-RT-2501). Also especial thanks to the Fundación AECC and Fundación Eugenio Rodriguez Pascual for supporting this work.

[Background]: Cutaneous squamous cell carcinoma (CSCC) is the second most frequent cancer in humans after basal cell carcinoma, and its incidence is dramatically rising. CSCC is rarely problematic, but given its high frequency, the absolute number of complicated cases is also high. It is necessary to identify molecular markers in order to recognize those CSCCs with poor prognosis. There is controversy concerning the role of epidermal growth factor receptor (EGFR) as a marker of prognosis in CSCC. In addition, EGFR-targeted therapies have emerged in recent years and a better understanding of the role of EGFR in CSCC may be of help for some patients in predicting prognosis and guiding curative management. [Objectives]: To evaluate the role of EGFR as a prognostic factor in CSCC. [Methods]: We evaluated clinical and histopathological features, including events of poor clinical evolution, in a series of 94 cases of CSCC. We also analysed EGFR expression by immunohistochemistry, fluorescent in situ hybridization and quantitative polymerase chain reaction. [Results]: We detected EGFR in 85 cases (90%), with overexpression in 33 cases (35%), and aberrant EGFR expression in the cytoplasm in 50 cases (53%). EGFR overexpression in the primary tumours was associated with lymph node progression, tumour–nodes–metastasis stage progression and proliferation (Ki-67 staining) in CSCC. EGFR overexpression and poor grade of differentiation were the strongest independent variables defining lymph node metastasis and progression in CSCC in a logistic regression model. [Conclusions]: We demonstrate that EGFR overexpression has prognostic implications associated with lymph node metastasis and progression in CSCC., J.P.‐L. was partially supported by FEDER and MICINN (PLE2009‐119, SAF2014‐56989‐R), Instituto de Salud Carlos III (PI07/0057, PI10/00328, PIE14/00066), Junta de Castilla y León (SAN673/SA26/08, SAN126/SA66/09, SA078A09, CSI034U13, BIO/SA31/15), IBSAL (IBY15/00003), the ‘Eugenio Rodríguez Pascual’, the ‘Fundación Inbiomed’ (Instituto Oncológico Obra Social de la Caja Guipozcoa‐San Sebastian, Kutxa) and the ‘Fundación Sandra Ibarra de Solidaridad frente al Cáncer’. C.R.‐C. is funded by Q3718001E (2009‐2010) and GRS 612/A/11 (2011‐2012) and ‘the Fundación Eugenio Rodríguez Pascual’. A.C.‐M. was supported by FIS (PI07/0057) and MICINN (PLE2009‐119).

Colorectal cancer (CRC) is the third most common cancer worldwide. The standard treatment in locally advanced rectal cancer is preoperative radiation alone or in combination with chemotherapy, followed by adjuvant chemotherapy. Rectal cancer is highly lethal, with only 20% of patients showing a complete remission (by RECIST) after standard treatment, although they commonly show local or systemic relapse likely due to its late detection and high chemotherapy resistance, among other reasons. Here, we explored the role of PAI1 (Serpin E1) in rectal cancer through the analyses of public patient databases, our own cohort of locally advanced rectal cancer patients and a panel of CRC cell lines. We showed that PAI1 expression is upregulated in rectal tumors, which is associated with decreased overall survival and increased metastasis and invasion in advanced rectal tumors. Accordingly, PAI1 expression is correlated with the expression of (Epithelial-to-Mesenchymal Transition) EMT-associated genes and genes encoding drug targets, including the tyrosine kinases PDGFRb, PDGFRa and FYN, the serine/threonine kinase PIM1 and BRAF. In addition, we demonstrate that cells expressing PAI1 protein are more sensitive to the PIM inhibitor AZD1208, suggesting that PAI1 could be used to predict response to treatment with PIM inhibitors and to complement radiotherapy in rectal tumors., This research was funded by Ministerio de Ciencia, Innovación y Universidades (MCIU) Plan Estatal de I+D+I 2018, Agencia Estatal de Investigación (AEI) and Regional Development European Funds (FEDER, European Union): RTI2018-097455-B-I00 (MCIU/AEI/FEDER, UE); grants from AEI-MICIU/FEDER (RED2018-102723-T; CIBER of Cancer (CB16/12/00275); Consejería de Salud of the Junta de Andalucía (PI-0397-2017); Consejeria of Economía, Conocimiento, Empresas y Universidad of the Junta de Andalucia (P18-RT-2501); Fundación AECC and Fundación Eugenio Rodriguez Pascual. SMG was funded by a Sara Borrell grant from ISCIII (CD16/00230) and the Fundación AECC.

[Background] Ovarian cancer is the leading cause of gynecologic cancer-related death, due in part to a late diagnosis and a high rate of recurrence. Primary and acquired platinum resistance is related to a low response probability to subsequent lines of treatment and to a poor survival. Therefore, a comprehensive understanding of the mechanisms that drive platinum resistance is urgently needed., [Methods] We used bioinformatics analysis of public databases and RT-qPCR to quantitate the relative gene expression profiles of ovarian tumors. Many of the dysregulated genes were cancer stem cell (CSC) factors, and we analyzed its relation to therapeutic resistance in human primary tumors. We also performed clustering and in vitro analyses of therapy cytotoxicity in tumorspheres., [Results] Using bioinformatics analysis, we identified transcriptional targets that are common endpoints of genetic alterations linked to platinum resistance in ovarian tumors. Most of these genes are grouped into 4 main clusters related to the CSC phenotype, including the DNA damage, Notch and C-KIT/MAPK/MEK pathways. The relative expression of these genes, either alone or in combination, is related to prognosis and provide a connection between platinum resistance and the CSC phenotype. However, the expression of the CSC-related markers was heterogeneous in the resistant tumors, most likely because there were different CSC pools. Furthermore, our in vitro results showed that the inhibition of the CSC-related targets lying at the intersection of the DNA damage, Notch and C-KIT/MAPK/MEK pathways sensitize CSC-enriched tumorspheres to platinum therapies, suggesting a new option for the treatment of patients with platinum-resistant ovarian cancer., [Conclusions] The current study presents a new approach to target the physiology of resistant ovarian tumor cells through the identification of core biomarkers. We hypothesize that the identified mutations confer platinum resistance by converging to activate a few pathways and to induce the expression of a few common, measurable and targetable essential genes. These pathways include the DNA damage, Notch and C-KIT/MAPK/MEK pathways. Finally, the combined inhibition of one of these pathways with platinum treatment increases the sensitivity of CSC-enriched tumorspheres to low doses of platinum, suggesting a new treatment for ovarian cancer., The authors thank the donors and the biobank of the Hospital Universitario Virgen del Rocío-Instituto de Biomedicina de Sevilla (Sevilla, Spain) (Andalusian Public Health System Biobank and ISCIII-Red de Biobancos PT17/0015/0041) for the human specimens used in this study. SM-G was supported by a Sara Borrell grant from ISCIII (Codigo CD16/00230) and BFA is funded by the Spanish Ministry of Education (FPU12/01380). AC lab was supported by grants from the Spanish Ministry of Economy and Competitivity, Plan Estatal de I + D + I 2013–2016, ISCIII (Fis: PI15/00045), the Ministry of Science, Innovation and Universities (RTI2018–097455-B-I00) and CIBER de Cáncer (CD16/12/00275), co-funded by FEDER from Regional Development European Funds (European Union), the AECC Foundation and Foundation Eugenio Rodriguez Pascual. Especial thanks to Consejeria de Salud of the Junta de Andalucia (PI-0397-2017) for supporting this work.

9 pags., 3 figs., 5 tabs., Background: Photodynamic therapy (PDT) or intense pulsed light (IPL) are efficient therapeutic methods in the treatment of superficial skin infections, and thus, they could be good options for onychomycosis treatment, the most common nail disorder. Methods: Forty patients, affected with different diagnosed types of onychomycosis in nails of the first toe, were randomly divided into two groups of 20 patients to be treated by PDT or IPL. Nail plates were softened with urea 40% by occlusive dressing for 12 hours during 3-7 days before treatments. Then, eight sessions separated by an interval of 2 weeks of a PDT protocol mediated by methylene blue (MB) and red laser diode (Periowave, λ = 670 nm, 200 mW) or an IPL protocol based on 10 pulses/cm (Dye-VL-F module, Alma Lasers, λ = 500-600 nm, 10 J) were applied. Results: Both treatments reduced significantly the Onychomycosis Severity Index (OSI) (P, Ministerio de Economía, Industria y Competitividad MINECO, Grant/Award Number: MAT 2017-83856-C3-1-P and MAT2015-68837-REDT; Fundación Eugenio Rodríguez Pascua

[Background]: Cutaneous squamous cell carcinoma (cSCC) represents the most common form of skin cancer after basal cell carcinoma, and can be both locally invasive and metastatic to distant sites. Growth rate (GR) has been poorly evaluated in cSCC, despite clinical evidence suggesting that GR is an important risk factor in cSCC., [Aim]: To analyse the influence of GR in cSCC prognosis., [Methods]: We retrospectively evaluated GR in a series of 90 cSCCs and tried to correlate GR with prognosis in cSCC., [Results]: We demonstrated that tumours with a GR of > 4 mm/month exhibit a higher risk of nodal progression and a shorter progression time to lymph node metastasis in cSCC than those with GR of < 4 mm/month. As expected, GR correlated with tumour proliferation, as determined by Ki-67 expression., [Conclusions]: We consider a GR of 4 mm/month as the cutoff point that distinguishes between rapid- and slow-progressing tumours and, more importantly, to identify a subset of high-risk cSCCs., JC was partially supported by Gerencia Regional de Salud, Junta de Castilla y León (GRS 1342/A/16) and by the programme INT/M/16/17. JPL was partially supported by FEDER and MICINN (SAF2014–56989-R), Instituto de Salud Carlos III (PIE14/00066), Junta de Castilla y León (BIO/SA31/15, CSI001416), IBSAL (IBY15/00003), the Eugenio Rodríguez Pascual, the Fundación Sandra Ibarra de Solidaridad frente al Cáncer and We can be Heroes Foundation. CR-C is funded by Q3718001E (2009–2010) and GRS 612/A/11 (2011–2012) and the Fundación Eugenio Rodríguez Pascual.

[Background]: Naevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by developmental alterations and multiple basal cell carcinomas. Mutations in PTCH1, which encodes a membrane receptor for Sonic Hedgehog, are associated with the development of the disease. Most of them produce a truncated protein, which is unable to suppress Smoothened protein and continuously activates the downstream pathway. [Objectives]: We aimed to characterize 22 unrelated Spanish patients with NBCCS, the largest cohort with Gorlin syndrome reported to date in Spain. [Methods]: Genomic analysis of PTCH1 was performed in patients with NBCCS and controls, and mutations were analysed using bioinformatics tools. [Results]: We report for the first time two young patients, one each with uterus didelphys and ganglioneuroma, within the context of NBCCS. One patient showing a severe phenotype of the disease had developed basal cell carcinomas since childhood. Sanger sequencing of PTCH1 in this cohort identified 17 novel truncating mutations (11 frameshift, five nonsense and one mutation affecting an exon–intron splice site) and two novel missense mutations that were predicted to be pathogenic. The patients showed great clinical variability and inconsistent genotype–phenotype correlation, as seen in relatives carrying similar mutations. [Conclusions]: This study contributes to increase the pool of clinical manifestations of NBCCS, as well as increasing the number of pathogenic mutations identified in PTCH1 predisposing to the condition. The inconsistencies found between phenotype and genotype suggest the involvement of other modifying factors, genetic, epigenetic or environmental., This project was funded by Fundación Rodríguez Pascual, Consejería de Sanidad de la Junta de Castilla y León‐FEDER (GRS165/A/07) and ISCIII‐FEDER (PI10/00219 and PI13/01741). The research at the Melanoma Unit in Barcelona was partially funded by grants 12/00840 from Fondo de Investigaciones Sanitarias, Spain; by the CIBER de Enfermedades Raras of the Instituto de Salud Carlos III, Spain; by the AGAUR 2014_SGR_603 of the Catalan Government, Spain; by a grant from ‘Fundació La Marató de TV3, 201331‐30’, Catalonia, Spain; and by the European Commission under the 6th Framework Programme, contract no. LSHC‐CT‐2006‐018702 (GenoMEL), under the 7th Framework Programme (Diagnoptics). J.C. is partially supported by grant GRS 1342/A/16 (Gerencia Regional de Salud de Castilla y León) and by the program INT/M/16/17 from the Gerencia Regional de Salud de Castilla y León.

[Background]: Cutaneous squamous cell carcinoma (CSCC) is the second most widespread cancer in humans and its incidence is rising. These tumours can evolve as diseases of poor prognosis, and therefore it is important to identify new markers to better predict its clinical evolution. [Objectives]: We aimed to identify the expression pattern of microRNAs (miRNAs or miRs) at different stages of skin cancer progression in a panel of murine skin cancer cell lines. Owing to the increasing importance of miRNAs in the pathogenesis of cancer, we considered the possibility that miRNAs could help to define the prognosis of CSCC and aimed to evaluate the potential use of miR-203 and miR-205 as biomarkers of prognosis in human tumours. [Methods]: Seventy-nine human primary CSCCs were collected at the University Hospital of Salamanca in Spain. We identified differential miRNA expression patterns at different stages of CSCC progression in a well-established panel of murine skin cancer cell lines, and then selected miR-205 and miR-203 to evaluate their association with the clinical prognosis and evolution of human CSCC. [Results]: miR-205 was expressed in tumours with pathological features recognized as indicators of poor prognosis such as desmoplasia, perineural invasion and infiltrative growth pattern. miR-205 was mainly expressed in undifferentiated areas and in the invasion front, and was associated with both local recurrence and the development of general clinical events of poor evolution. miR-205 expression was an independent variable selected to predict events of poor clinical evolution using the multinomial logistic regression model described in this study. In contrast, miR-203 was mainly expressed in tumours exhibiting the characteristics associated with a good prognosis, was mainly present in well-differentiated zones, and rarely expressed in the invasion front. Therefore, the expression and associations of miR-205 and miR-203 were mostly mutually exclusive. Finally, using a logistic biplot we identified three clusters of patients with differential prognosis based on miR-203 and miR-205 expression, and pathological tumour features. [Conclusions]: miR-205 and miR-203 tended to exhibit mutually exclusive expression patterns in human CSCC. This work highlights the utility of miR-205 and miR-203 as prognostic markers in CSCC., J.P.-L. was partially supported by FEDER and MICINN (PLE2009-119, SAF2014-56989-R), Instituto de Salud Carlos III (PI07/0057, PI10/00328, PIE14/00066), Junta de Castilla y Leon (SA078A09, CSI034U13, CSI001U16), the ‘Eugenio Rodriguez Pascual’, the ‘Fundacion Inbiomed’ (Instituto Oncologico Obra Social de la Caja Guipozcoa-San Sebastian, Kutxa), IBSAL (IBY15/00003) and the ‘Fundacion Sandra Ibarra de Solidaridad frente al Cancer’. C.R.-C. is funded by Q3718001E (2009–2010) y GRS 612/A/11 (2011–2012) and the ‘Fundacion Eugenio Rodriguez Pascual’. A.C.-M. was supported by FIS (PI07/0057) and MICINN (PLE2009-119). J.H.M. was supported by the National Institutes of Health, a National Cancer Institute grant (R01 CA116481) and the Low-Dose Scientific Focus Area, Office of Biological & Environmental Research, U.S. Department of Energy (DE-AC02-05CH11231). J.C. was partially supported by Gerencia Regional de Salud (Junta de Castilla y Leon), GRS 1342 / A / 16.

The liver displays a remarkable regenerative capacity triggered upon tissue injury or resection. However, liver regeneration can be overwhelmed by excessive parenchymal destruction or diminished by pre-existing conditions hampering repair. Fibroblast growth factor 19 (FGF19, rodent FGF15) is an enterokine that regulates liver bile acid and lipid metabolism, and stimulates hepatocellular protein synthesis and proliferation. FGF19/15 is also important for liver regeneration after partial hepatectomy (PH). Therefore recombinant FGF19 would be an ideal molecule to stimulate liver regeneration, but its applicability may be curtailed by its short half-life. We developed a chimaeric molecule termed Fibapo in which FGF19 is covalently coupled to apolipoprotein A-I. Fibapo retains FGF19 biological activities but has significantly increased half-life and hepatotropism. Here we evaluated the proregenerative activity of Fibapo in two clinically relevant models where liver regeneration may be impaired: acetaminophen (APAP) poisoning, and PH in aged mice. The only approved therapy for APAP intoxication is N-acetylcysteine (NAC) and no drugs are available to stimulate liver regeneration. We demonstrate that Fibapo reduced liver injury and boosted regeneration in APAPintoxicated mice. Fibapo improved survival of APAP-poisoned mice when given at later time points, when NAC is ineffective. Mechanistically, Fibapo accelerated recovery of hepatic glutathione levels, potentiated cell growth-related pathways and increased functional liver mass. When Fibapo was administered to old mice prior to PH, liver regeneration was markedly increased. The exacerbated injury developing in these mice upon PH was attenuated, and the hepatic biosynthetic capacity was enhanced. Fibapo reversed metabolic and molecular alterations that impede regeneration in aged livers. It reduced liver steatosis and downregulated p21 and hepatocyte nuclear factor 4 α (Hnf4α) levels, whereas it stimulated Foxm1b gene expression. Together our findings indicate that FGF19 variants retaining the metabolic and growth-promoting effects of this enterokine may be valuable for the stimulation of liver regeneration., Work in the authors' laboratory is supported by CIBERehd and Grants from Instituto de Salud Carlos III (ISCIII) co-financed by 'Fondo Europeo de Desarrollo Regional' (FEDER) 'Una manera de hacer Europa', numbers: FIS PI13/00359, PI13/00385 and PI16/01126. Grants SAF2015-66515-R, SAF201569944-R, SAF 2016-75972 R from Ministerio de Economía y Competitividad, and the center grant P50AA011999 funded by NIAAA. 'Ramón y Cajal-I3' contract to MUL, Mineco-FPI Fellowship to MB-V. Marie Curie EU contract to MGF-B. Fundación M Torres; Fundación Eugenio Rodríguez Pascual; Fundación Mario Losantos; Fundación Familia Puig-Infante and Fundación Bancaria La CaixaHepacare Project.

Diseases of complex origin have a component of quantitative genetics that contributes to their susceptibility and phenotypic variability. However, after several studies, a major part of the genetic component of complex phenotypes has still not been found, a situation known as “missing heritability.” Although there have been many hypotheses put forward to explain the reasons for the missing heritability, its definitive causes remain unknown. Complex diseases are caused by multiple intermediate phenotypes involved in their pathogenesis and, very often, each one of these intermediate phenotypes also has a component of quantitative inheritance. Here we propose that at least part of the missing heritability can be explained by the genetic component of intermediate phenotypes that is not detectable at the level of the main complex trait. At the same time, the identification of the genetic component of intermediate phenotypes provides an opportunity to identify part of the missing heritability of complex diseases., JPL was partially supported by FEDER and MICINN (PLE2009-119, SAF2014-56989-R), Instituto de Salud Carlos III (PI07/0057, PI10/00328, PIE14/00066), Junta de Castilla y León (CSI034U13, BIO/SA31/15), “Proyectos integrados IBSAL 2015” (IBY15/00003), the Eugenio Rodriguez Pascual”, the “Fundacion Inbiomed” (Instituto Oncologico Obra Social de la Caja Guipozcoa-San Sebastian, Kutxa), and the “Fundacion Sandra Ibarra de Solidaridad frente al Cancer”. CR-C is funded by Q3718001E (2009–2010) y GRS 612/A/11 (2011–2012) and “the Fundacion Eugenio Rodrıguez Pascual”. AC was supported by FIS (PI07/0057) and MICINN (PLE2009-119). SCLL was funded by a JAEdoc Fellowship (CSIC)/FSE. JHM was supported by the National Institutes of Health, a National Cancer Institute grant (R01 CA116481), and the Low-Dose Scientific Focus Area, Office of Biological & Environmental Research, US Department of Energy (DE-AC02-05CH11231).

PMCID: PMC3388112.-- et al., The Aurora-A kinase gene is frequently amplified and/or overexpressed in a variety of human cancers, leading to major efforts to develop therapeutic agents targeting this pathway. Here, we show that Aurora-A is targeted for ubiquitination and subsequent degradation by the F-box protein FBXW7 in a process that is regulated by GSK3β. Using a series of truncated Aurora-A proteins and site-directed mutagenesis, we identified distinct FBXW7 and GSK3β-binding sites in Aurora-A. Mutation of critical residues in either site substantially disrupts degradation of Aurora-A. Furthermore, we show that loss of Pten results in the stabilization of Aurora-A by attenuating FBXW7-dependent degradation of Aurora-A through the AKT/GSK3β pathway. Moreover, radiation-induced tumor latency is significantly shortened in Fbxw7 +/-Pten +/- mice as compared with either Fbxw7 +/- or Pten +/- mice, indicating that Fbxw7 and Pten appear to cooperate in suppressing tumorigenesis. Our results establish a novel posttranslational regulatory network in which the Pten and Fbxw7 pathways appear to converge on the regulation of Aurora-A level. ©2012 AACR., J.-H. Mao was supported by the NIH, National Cancer Institute grant R01 CA116481, the Low Dose Scientific Focus Area, Office of Biological and Environmental Research, of the U.S. Department of Energy under contract no. DEAC02- 05CH11231, and Laboratory Directed Research and Development Program (LDRD). A. Balmain was supported by the Department of Energy Low Dose Radiation Research Program DESC0003679 and the National Cancer Institute grant U01 CA141455. J. Losada-Losada was partially supported by FEDER, MICINN (PLE2009-119), FIS (PI10/00328), and "Fundación Eugenio Rodríguez- Pascual."

New subventricular zone (SVZ)-derived neuroblasts that migrate via the rostral migratory stream are continuously added to the olfactory bulb (OB) of the adult rodent brain. Anosmin-1 (A1) is an extracellular matrix protein that binds to FGF receptor 1 (FGFR1) to exert its biological effects. When mutated as in Kallmann syndrome patients, A1 is associated with severe OB morphogenesis defects leading to anosmia and hypogonadotropic hypogonadism. Here, we show that A1 over-expression in adult mice strongly increases proliferation in the SVZ, mainly with symmetrical divisions, and produces substantial morphological changes in the normal SVZ architecture, where we also report the presence of FGFR1 in almost all SVZ cells. Interestingly, for the first time we show FGFR1 expression in the basal body of primary cilia in neural progenitor cells. Additionally, we have found that A1 over-expression also enhances neuroblast motility, mainly through FGFR1 activity. Together, these changes lead to a selective increase in several GABAergic interneuron populations in different OB layers. These specific alterations in the OB would be sufficient to disrupt the normal processing of sensory information and consequently alter olfactory memory. In summary, this work shows that FGFR1-mediated A1 activity plays a crucial role in the continuous remodelling of the adult OB., This research was supported by grants from the Spanish Ministerio de Economía, Innovación y Competitividad MINECO (SAF2009-07842, ADE10-0010, RD07-0060-2007, RD12-0032-12 and SAF2012-40023 to FdC; and BFU2010-18284 to JMG-V), FISCAM (Gobierno de Castilla-La Mancha, Spain—Grant Number PI2007-66), the Junta de Castilla y León (Spain, to EW), and from the Fundación Eugenio Rodríguez Pascual (Spain) to FdC. DGG and VMB were PhD students hired by Gobierno de Castilla-La Mancha (MOV2010-JI/11 and MOV2007-JI/19, respectively). FdCS is a CSIC staff scientist in special permission hired by SESCAM (Gobierno de Castilla-La Mancha, Spain). PFE was a researcher hired by SESCAM (Gobierno de Castilla-La Mancha) and ADE10-0010.

During development of the central nervous system, anosmin-1 (A1) works as a chemotropic cue contributing to axonal outgrowth and collateralization, as well as modulating the migration of different cell types, fibroblast growth factor receptor 1 (FGFR1) being the main receptor involved in all these events. To further understand the role of A1 during development, we have analysed the over-expression of human A1 in a transgenic mouse line. Compared with control mice during development and in early adulthood, A1 over-expressing transgenic mice showed an enhanced oligodendrocyte precursor cell (OPC) proliferation and a higher number of OPCs in the subventricular zone and in the corpus callosum (CC). The migratory capacity of OPCs from the transgenic mice is increased in vitro due to a higher basal activation of ERK1/2 mediated through FGFR1 and they also produced more myelin basic protein (MBP). In vivo, the over-expression of A1 resulted in an elevated number of mature oligodendrocytes with higher levels of MBP mRNA and protein, as well as increased levels of activation of the ERK1/2 proteins, while electron microscopy revealed thicker myelin sheaths around the axons of the CC in adulthood. Also in the mature CC, the nodes of Ranvier were significantly longer and the conduction velocity of the nerve impulse in vivo was significantly increased in the CC of A1 over-expressing transgenic mice. Altogether, these data confirmed the involvement of A1 in oligodendrogliogenesis and its relevance for myelination., This work was supported by grants from the Spanish Ministerio de Economía y Competitividad-MINECO (Grant Numbers SAF2009-07842, SAF2012-40023, RD07-0060-2007 and RD12-0032-12 Red Española de Esclerosis Múltple) and the “Fundación Eugenio Rodríguez Pascual” to FdC; the Fundación para la Investigación Socio-sanitaria de Castilla La-Mancha FISCAM (Grant Number PI2007-66 to FdC and PI2009-29 to PFE); from the Spanish MINECO (Grant Numbers BFU2008-00899 and BFU2008-03390) and the Junta de Andalucía (Grant Numbers BIO-122, CVI-02487 and P07-CVI-02686) to JMDG and AGM; and from the Spanish MINECO (Grant Number BFU-2009-08404) to RL. VMB was hired under FISCAM (MOV2007-JI/19), SAF2009-07842 and RD07-0060-2007; PFE was hired by the Servicio de Salud de Castilla La-Mancha (SESCAM, Gobierno de Castilla-La Mancha) and under grant ADE10-0010 from MINECO to FdC. FdC is a CSIC Staff Scientist on leave of absence and currently hired by SESCAM.

Metastatic dissemination is the most frequent cause of death of sporadic colorectal cancer (sCRC) patients. Genomic abnormalities which are potentially characteristic of such advanced stages of the disease are complex and so far, they have been poorly described and only partially understood. We evaluated the molecular heterogeneity of sCRC tumors based on simultaneous assessment of the overall GEP of both coding mRNA and non-coding RNA genes in primary sCRC tumor samples from 23 consecutive patients and their paired liver metastases. Liver metastases from the sCRC patients analyzed, systematically showed deregulated transcripts of those genes identified as also deregulated in their paired primary colorectal carcinomas. However, some transcripts were found to be specifically deregulated in liver metastases (vs. non-tumoral colorectal tissues) while expressed at normal levels in their primary tumors, reflecting either an increased genomic instability of metastatic cells or theiradaption to the liver microenvironment. Newly deregulated metastatic transcripts included overexpression of APOA1, HRG, UGT2B4, RBP4 and ADH4 mRNAS and the miR-3180-3p, miR-3197, miR-3178, miR-4793 and miR-4440 miRNAs, together with decreased expression of the IGKV1-39, IGKC, IGKV1-27, FABP4 and MYLK mRNAS and the miR-363, miR-1, miR-143, miR-27b and miR-28-5p miRNAs. Canonical pathways found to be specifically deregulated in liver metastatic samples included multiple genes related with intercellular adhesion and the metastatic processes (e.g., IGF1R, PIK3CA, PTEN and EGFR), endocytosis (e.g., the PDGFRA, SMAD2, ERBB3, PML and FGFR2), and the cell cycle (e.g., SMAD2, CCND2, E2F5 and MYC). Our results also highlighted the activation of genes associated with the TGFβ signaling pathway, -e.g. RHOA, SMAD2, SMAD4, SMAD5, SMAD6, BMPR1A, SMAD7 and MYC-, which thereby emerge as candidate genes to play an important role in CRC tumor metastasis., This work has been partially supported by grants from the Instituto de Salud Carlos III (ISCIII; Ministerio de Sanidad y Consumo, Madrid, Spain) (PI12/02053-FIS), Gerencia Regional de Salud de Castilla y León, Valladolid, Spain (GRS1302/A/16), Consejería de Sanidad (Junta de Castilla y Leon, Valladolid, Spain) (BIO/SA02/13 and BIO/SA46/14), RTICC from the ISCIII (RD12/0020/0035-FEDER, RD12/0036/0048-FEDER), Fundación Memoria de Don Samuel Solórzano Barruso, (Salamanca, Spain) and Fundación Eugenio Rodríguez Pascual, (Madrid, Spain). JM Sayagués and ME Sarasquete are supported by grants (CES11/004 and CP13/00080; respectively) from the ISCIII, Ministerio de Ciencia e Innovación, Madrid, Spain.

Different data support a role for the epidermal growth factor receptor (EGFR) pathway during liver regeneration and hepatocarcinogenesis. However, important issues, such as the precise mechanisms mediating its actions and the unique versus redundant functions, have not been fully defined. Here, we present a novel transgenic mouse model expressing a hepatocyte-specific truncated form of human EGFR, which acts as negative dominant mutant (ΔEGFR) and allows definition of its tyrosine kinase-dependent functions. Results indicate a critical role for EGFR catalytic activity during the early stages of liver regeneration. Thus, after two-thirds partial hepatectomy, ΔEGFR livers displayed lower and delayed proliferation and lower activation of proliferative signals, which correlated with overactivation of the transforming growth factor-β pathway. Altered regenerative response was associated with amplification of cytostatic effects of transforming growth factor-β through induction of cell cycle negative regulators. Interestingly, lipid synthesis was severely inhibited in ΔEGFR livers after partial hepatectomy, revealing a new function for EGFR kinase activity as a lipid metabolism regulator in regenerating hepatocytes. In spite of these profound alterations, ΔEGFR livers were able to recover liver mass by overactivating compensatory signals, such as c-Met. Our results also indicate that EGFR catalytic activity is critical in the early preneoplastic stages of the liver because ΔEGFR mice showed a delay in the appearance of diethyl-nitrosamine-induced tumors, which correlated with decreased proliferation and delay in the diethyl-nitrosamine-induced inflammatory process. Conclusion: These studies demonstrate that EGFR catalytic activity is critical during the initial phases of both liver regeneration and carcinogenesis and provide key mechanistic insights into how this kinase acts to regulate liver pathophysiology. (Hepatology 2016;63:604-619), Cofunded by the Ministry of Economy and Competitiveness-MINECO and the European Regional Development Fund-FEDER, Spain (Contract grant numbers: BFU2012-35538 and ISCIII-RTICC RD12-0036-0029 to I.F.-IDIBELL; SAF2009-12477 to A.S.-UCM/IdISSC; RETICS-RD12/0019/0023 and SAF2011-30526-C02-01 to J.-C.S.-CIEMAT/CIBERER/IIS-FJD; SAF2012-33283 to A.M.V.-CSIC/CIBERDEM). People Programme (Marie Curie Actions) of the FP7-2012, under REA grant agreement #PITN-GA-2012-316549 (IT-LIVER) to I.F.-IDIBELL and to A.S.-UCM/IdISSC. AGAUR-Generalitat de Catalunya to I.F-IDIBELL (Contract grant number: 2009SGR-312). Dirección General de Investigación de la Comunidad de Madrid, Spain (Contract grant number S2010/BMD-2402 to A.S.-UCM/IdISSC). Fundación Eugenio Rodríguez Pascual to M.G.-B.-CIEMAT/CIBERER/IIS-FJD. J.L.-L. was recipient of a predoctoral fellowship from IDIBELL; D.C.-D. and A.M.-P. were recipients of predoctoral fellowships from MINECO, Spain (Contract grant number: FPI - BES-2013-064609 and BES-2007-16187, respectively).

The T-cell receptor β (TCRβ)/pre-TCRα (pTα) pre-TCR complex (pre-TCR) signals the expansion and differentiation of de-veloping thymocytes. Functional pro-perties of the pre-TCR rely on its unique pTα chain, which suggests the participation of specific intracellular adaptors. However, pTα-interacting molecules remain unknown. Here, we identified a polyproline-arginine sequence in the human pTα cytoplasmic tail that interacted in vitro with SH3 domains of the CIN85/CMS family of adaptors, and mediated the recruitment of multiprotein complexes involving all (CMS, CIN85, and CD2BP3) members. Supporting the physiologic relevance of this interaction, we found that 1 such adaptor, CMS, interacted in vivo with human pTα, and its expression was selectively up-regulated during human thymopoiesis in pre-TCR–activated thymocytes. Upon activation, pre-TCR clustering was induced, and CMS and polymerized actin were simultaneously recruited to the pre-TCR activation site. CMS also associated via its C-terminal region to the actin cytoskeleton in the endocytic compartment, where it colocalized with internalized pTα in traffic to lysosomal degradation. Notably, deletion of the pTα CIN85/CMS-binding motif impaired pre-TCR–mediated Ca2+ mobilization and NFAT transcriptional activity, and precluded activation induced by overexpression of a CMS-SH3 N-terminal mutant. These results provide the first molecular evidence for a pTα intracellular adaptor involved in pre-TCR function., This work was supported in part by grants from the Plan Nacional de Biomedicina (SAF2004-01122), Comunidad de Madrid (GR/SAL/0143/2004, S-SAL0304-2006), Fundacio´n la Caixa (ON03/109-00), and Fundacio´n Rodrı´guez Pascual, and by an institutional grant from the Fundacio´n Ramo´n Areces. G.N. and P.F. were supported by the Plan Nacional de Biomedicina SAF2001- 1269 and SAF2004-01122 grants, respectively; M.N.N. was supported by a Formacio´n Personal Investigador (FPI) fellowship.

PMCID: PMC4560637, Breast cancer is a major cause of mortality in women. The transcription factor SNAI2 has been implicated in the pathogenesis of several types of cancer, including breast cancer of basal origin. Here we show that SNAI2 is also important in the development of breast cancer of luminal origin in MMTV-ErbB2 mice. SNAI2 deficiency leads to longer latency and fewer luminal tumors, both of these being characteristics of pretumoral origin. These effects were associated with reduced proliferation and a decreased ability to generate mammospheres in normal mammary glands. However, the capacity to metastasize was not modified. Under conditions of increased ERBB2 oncogenic activity after pregnancy plus SNAI2 deficiency, both pretumoral defects - latency and tumor load - were compensated. However, the incidence of lung metastases was dramatically reduced. Furthermore, SNAI2 was required for proper postlactational involution of the breast. At 3 days post lactational involution, the mammary glands of Snai2-deficient mice exhibited lower levels of pSTAT3 and higher levels of pAKT1, resulting in decreased apoptosis. Abundant noninvoluted ducts were still present at 30 days post lactation, with a greater number of residual ERBB2+ cells. These results suggest that this defect in involution leads to an increase in the number of susceptible target cells for transformation, to the recovery of the capacity to generate mammospheres and to an increase in the number of tumors. Our work demonstrates the participation of SNAI2 in the pathogenesis of luminal breast cancer, and reveals an unexpected connection between the processes of postlactational involution and breast tumorigenesis in Snai2-null mutant mice., JPL was partially supported by FEDER and MICINN (PLE2009-119, SAF2014-56989-R), Instituto de Salud Carlos III (PI07/0057, PI10/00328, PIE14/00066), Junta de Castilla y León (SAN673/SA26/08, SAN126/SA66/09, SA078A09, CSI034U13), the “Fundación Eugenio Rodríguez Pascual”, the “Fundación Inbiomed” (Instituto Oncológico Obra Social de la Caja Guipozcoa-San Sebastian, Kutxa), and the “Fundación Sandra Ibarra de Solidaridad frente al Cáncer”. AC was supported by FIS (PI07/0057) and MICINN (PLE2009-119). SCLL was funded by a JAEdoc Fellowship (CSIC)/FSE. MMSF and ABG are funded by fellowships from the Junta de Castilla y Leon. JHM was supported by the National Institutes of Health, a National Cancer Institute grant (R01 CA116481), and the Low-Dose Scientific Focus Area, Office of Biological & Environmental Research, US Department of Energy (DE-AC02-05CH11231).

Article available at http://www.jimmunol.org/cgi/content/abstract/177/6/3711, Notch signaling is critical for T cell development of multipotent hemopoietic progenitors. Yet, how Notch regulates T cell fate specification during early thymopoiesis remains unclear. In this study, we have identified an early subset of CD34highc-kit+flt3+IL-7R+ cells in the human postnatal thymus, which includes primitive progenitors with combined lymphomyeloid potential. To assess the impact of Notch signaling in early T cell development, we expressed constitutively active Notch1 in such thymic lymphomyeloid precursors (TLMPs), or triggered their endogenous Notch pathway in the OP9-Delta-like1 stroma coculture. Our results show that proliferation vs differentiation is a critical decision influenced by Notch at the TLMP stage. We found that Notch signaling plays a prominent role in inhibiting non-T cell differentiation (i.e., macrophages, dendritic cells, and NK cells) of TLMPs, while sustaining the proliferation of undifferentiated thymocytes with T cell potential in response to unique IL-7 signals. However, Notch activation is not sufficient for inducing T-lineage progression of proliferating progenitors. Rather, stroma-derived signals are concurrently required. Moreover, while ectopic IL-7R expression cannot replace Notch for the maintenance and expansion of undifferentiated thymocytes, Notch signals sustain IL-7R expression in proliferating thymocytes and induce IL-7R up-regulation in a T cell line. Thus, IL-7R and Notch pathways cooperate to synchronize cell proliferation and suppression of non-T lineage choices in primitive intrathymic progenitors, which will be allowed to progress along the T cell pathway only upon interaction with an inductive stromal microenvironment. These data provide insight into a mechanism of Notch-regulated amplification of the intrathymic pool of early human T cell progenitors, This work was supported by grants from Plan Nacional de Biomedicina (SAF2004-01122 and GEN2003-20649-C06-02), Comunidad de Madrid (GR/SAL/0143/ 2004), Fundación La Caixa (ON03/109-00), and Fundación Eugenio Rodríguez Pascual. We thank the Fundación Ramón Areces for an institutional grant to the Centro de Biología Molecular Severo Ochoa

The present study tests the hypothesis that higher consumption of bakery products, sweetened soft drinks and yogurt is associated with higher intake of energy, saturated fats, sugars and worse overall diet quality among Spanish children. This is a cross-sectional study covering 1112 children aged 6.0–7.0 years in four Spanish cities. Nutrient and food intake were obtained through a food-frequency questionnaire, and overall diet quality calculated using the healthy-eating index (HEI) developed by Kennedyet al.(1995). Standardized methods were used to measure anthropometric variables. Associations of interest were summarized as the difference in nutrient and food consumption between the value of the fifth and the first quintile of consumption (dq) of bakery products, sweetened soft drinks or yogurt, adjusted for energy intake and BMI. Bakery products, sweetened soft drinks and yogurt supplied 15·5, 1·0 and 5·6 % energy intake respectively. Higher consumption of these three foods was associated with greater energy intake (PPPPPPPP

The aim of the present study was to assess the principal food sources of energy and nutrients among Spanish children. We used a cross-sectional study design, based on results obtained from a food-frequency questionnaire. The sample included 1112 children, aged 6-7 years, from Cadiz, Madrid, Orense and Murcia, Spain. Children were selected through random cluster-sampling in schools. We analysed the percentage contributed by each food item to total energy and nutrient intake. The most important food sources were: white bread in the case of carbohydrate (13.4 %); olive oil in the case of total lipids (18.3 %) and monounsaturated fatty acids (29.2 %); whole milk in the case of protein (10.2 %) and saturated fatty acids (14.9 %); chips (French fried potatoes) in the case of polyunsaturated fatty acids (30.4 %). The greatest proportion of Na, consumed in excess, came from salt added to meals. Ham ranked second as a source of saturated fats. Fruits and green leafy vegetables proved to have great relevance as sources of fibre and vitamins, though with regard to the latter, it was observed that fortified foods (breakfast cereals, dairy products, fruit juices, etc.) had come to play a relevant role in many cases. In conclusion, the nutritional profile of Spanish school-aged children aged 6-7 years could be improved by nutritional policies targeted at limiting their consumption of ham (cured or cooked) and of salt added to meals, replacing whole milk with semi-skimmed milk, encouraging the consumption of products rich in complex carbohydrates already present in children's diets (bread, pasta, rice) and promoting less fatty ways of cooking food. This study was partly funded by grants from the Inter-national Olive Oil Board (Consejo Oleı ́cola Internacional),Madrid Regional Authority (Comunidad de Madrid), PedroBarrie ́de la Maza Foundation and Eugenio Rodrı ́guezPascual Foundation Sí

¿-Tocopheryl succinate (¿-TOS) is a well-known mitochondrially targeted anticancer compound, however, it is highly hydrophobic and toxic. In order to improve its activity and reduce its toxicity, new surfactant-free biologically active nanoparticles (NP) were synthesized. A methacrylic derivative of ¿-TOS (MTOS) was prepared and incorporated in amphiphilic pseudoblock copolymers when copolymerized with N-vinylpyrrolidone (VP) by free radical polymerization (poly(VP-co-MTOS)). The selected poly(VP-co-MTOS) copolymers formed surfactant-free NP by nanoprecipitation with sizes between 96 and 220 nm and narrow size distribution, and the in vitro biological activity was tested. In order to understand the structure-activity relationship three other methacrylic monomers were synthesized and characterized: MVE did not have the succinate group, SPHY did not have the chromanol ring, and MPHY did not have both the succinate group and the chromanol ring. The corresponding families of copolymers (poly(VP-co-MVE), poly(VP-co-SPHY), and poly(VP-co-MPHY)) were synthesized and characterized, and their biological activity was compared to poly(VP-co-MTOS). Both poly(VP-co-MTOS) and poly(VP-co-MVE) presented triple action: reduced cell viability of cancer cells with little or no harm to normal cells (anticancer), reduced viability of proliferating endothelial cells with little or no harm to quiescent endothelial cells (antiangiogenic), and efficiently encapsulated hydrophobic molecules (nanocarrier). The anticancer and antiangiogenic activity of the synthesized copolymers is demonstrated as the active compound (vitamin E or ¿-tocopheryl succinate) do not need to be cleaved to trigger the biological action targeting ubiquinone binding sites of complex II. Poly(VP-co-SPHY) and poly(VP-co-MPHY) also formed surfactant-free NP that were also endocyted by the assayed cells; however, these NP did not selectively reduce cell viability of cancer cells. Therefore, the chromanol ring of the vitamin E analogues has an important role in the biological activity of the copolymers. Moreover, when succinate moiety is substituted and vitamin E is directly linked to the macromolecular chain through an ester bond, the biological activity is maintained., Authors acknowledge Dr. Gema Rodriǵ uez, Lautaro Biancotto, David Goḿ ez, and Rosa Ana Ramiŕ ez for their help in AFM, SEM/TEM, and cell culture experiments. Authors also thank Dr. M.P. Murphy for providing MitoQ for the experiments. This work was funded by the Spanish Ministry of Economy and Competitiveness (MAT2010−18155), Eugenio Rodriǵ uez Pascual Foundation, CIBER BBN-ECO Foundation project, and the European University of Madrid.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al., [Background]: An essential question in cancer is why individuals with the same disease have different clinical outcomes. Progress toward a more personalized medicine in cancer patients requires taking into account the underlying heterogeneity at different molecular levels. [Results]: Here, we present a model in which there are complex interactions at different cellular and systemic levels that account for the heterogeneity of susceptibility to and evolution of ERBB2-positive breast cancers. Our model is based on our analyses of a cohort of mice that are characterized by heterogeneous susceptibility to ERBB2-positive breast cancers. Our analysis reveals that there are similarities between ERBB2 tumors in humans and those of backcross mice at clinical, genomic, expression, and signaling levels. We also show that mice that have tumors with intrinsically high levels of active AKT and ERK are more resistant to tumor metastasis. Our findings suggest for the first time that a site-specific phosphorylation at the serine 473 residue of AKT1 modifies the capacity for tumors to disseminate. Finally, we present two predictive models that can explain the heterogeneous behavior of the disease in the mouse population when we consider simultaneously certain genetic markers, liver cell signaling and serum biomarkers that are identified before the onset of the disease. [Conclusions]: Considering simultaneously tumor pathophenotypes and several molecular levels, we show the heterogeneous behavior of ERBB2-positive breast cancer in terms of disease progression. This and similar studies should help to better understand disease variability in patient populations., JPL was partially supported by FEDER and MICINN (PLE2009-119), FIS (PI07/0057, PI10/00328, PIE14/00066), the Junta de Castilla y León (SAN673/SA26/08; SAN126/SA66/09, SA078A09, CSI034U13), the “Fundación Eugenio Rodríguez Pascual”, the Fundación Inbiomed (Instituto Oncológico Obra Social de la Caja Guipozcoa-San Sebastian, Kutxa), and the “Fundación Sandra Ibarra de Solidaridad frente al Cáncer”. AC was supported by MICINN (PLE2009-119). SCLL is funded by a JAEdoc Fellowship (CSIC)/FSE. MMSF and ABG are funded by fellowships from the Junta de Castilla y Leon. WR was supported by a Forschungsstipendium of the Deutsche Forschungsgemeinschaft (DFG) [RE 3108/1-1]. TN, BPB and DYL acknowledge support from the US Department of Energy Low-Dose SFA Program at Berkeley Lab [DE-AC02-05CH11231], the National Institutes of Health [RC1NS069177] and the California Breast Cancer Research Program [15IB-0063]. JHM was supported by the National Institutes of Health, a National Cancer Institute grant (R01 CA116481), and the Low-Dose Scientific Focus Area, Office of Biological and Environmental Research, US Department of Energy (DE-AC02-05CH11231).

This work reports the molecular genetic study of a patient who suffered from Glanzmann thrombasthenia (GT). Structural analysis of the glycoprotein (GP) IIb and GPIIIa genes showed the presence of a homozygous G1846→T transversion in exon 11 of GPIIIa that changes Glu616→Stop. Cytometric and immunochemical analysis indicated that platelet GPIIb-IIIa was absent in the proband but present at normal levels in the heterozygous relatives. The following observations indicate that this mutation is responsible for the thrombasthenic phenotype of the proband. (1) We failed to detect mutations other than [T1846]GPIIIa in the coding region of both GPIIb and GPIIIa genes. (2) The G1846→T mutation was observed in either parent and a brother of the proband, but none of 100 unrelated individuals carried this defect. (3) Pulse-chase and immunoprecipitation analysis of GPIIb-IIIa complexes in cells transiently cotransfected with cDNAs encoding normal GPIIb and [T1846]GPIIIa showed neither maturation of GPIIb nor complex formation and surface exposure of GPIIb-ΔGPIIIa. These observations indicate that the sequence from Glu616 to Thr762 in GPIIIa is essential for heterodimerization with GPIIb. Polymerase chain reaction-based analysis demonstrated the presence of normal levels of full-length GPIIIa-mRNA in the proband and in heterozygous relatives. In addition, a shortened transcript, with a 324-nucleotide deletion, resulting from inframe skipping of exons 10 and 11, was detectable upon reamplification of the DNA. Thus, unlike other nonsense mutations, [T1846]GPIIIa does not lead to abnormal processing or reduction in the number of transcripts with the termination codon., Supported in part by grants from Dirección General de Investigación Científica y Técnica (PB94-1544), Fondo de Investigaciones Sanitarias (96/2014), CAM: CO7191, and European Community concerted action contract BMH1-CT93-1685. M.F. was the recipient of a predoctoral fellowship from the Comunidad Autónoma de Madrid. C.G.M. was recipient of a grant from Fundación Rodríguez Pascual.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al., [Introduction]: Endocrine therapies targeting cell proliferation and survival mediated by estrogen receptor α (ERα) are among the most effective systemic treatments for ERα-positive breast cancer. However, most tumors initially responsive to these therapies acquire resistance through mechanisms that involve ERα transcriptional regulatory plasticity. Herein we identify VAV3 as a critical component in this process. [Methods]: A cell-based chemical compound screen was carried out to identify therapeutic strategies against resistance to endocrine therapy. Binding to ERα was evaluated by molecular docking analyses, an agonist fluoligand assay and short hairpin (sh)RNA-mediated protein depletion. Microarray analyses were performed to identify altered gene expression. Western blot analysis of signaling and proliferation markers, and shRNA-mediated protein depletion in viability and clonogenic assays, were performed to delineate the role of VAV3. Genetic variation in VAV3 was assessed for association with the response to tamoxifen. Immunohistochemical analyses of VAV3 were carried out to determine its association with therapeutic response and different tumor markers. An analysis of gene expression association with drug sensitivity was carried out to identify a potential therapeutic approach based on differential VAV3 expression. [Results]: The compound YC-1 was found to comparatively reduce the viability of cell models of acquired resistance. This effect was probably not due to activation of its canonical target (soluble guanylyl cyclase), but instead was likely a result of binding to ERα. VAV3 was selectively reduced upon exposure to YC-1 or ERα depletion, and, accordingly, VAV3 depletion comparatively reduced the viability of cell models of acquired resistance. In the clinical scenario, germline variation in VAV3 was associated with the response to tamoxifen in Japanese breast cancer patients (rs10494071 combined P value = 8.4 × 10-4). The allele association combined with gene expression analyses indicated that low VAV3 expression predicts better clinical outcome. Conversely, high nuclear VAV3 expression in tumor cells was associated with poorer endocrine therapy response. Based on VAV3 expression levels and the response to erlotinib in cancer cell lines, targeting EGFR signaling may be a promising therapeutic strategy. [Conclusions]: This study proposes VAV3 as a biomarker and a rationale for its use as a signaling target to prevent and/or overcome resistance to endocrine therapy in breast cancer., This work was supported by grants from the Eugenio Rodríguez Pascual Foundation (2012, to MAP), the Government of Catalonia (2009-SGR283, to AV and MAP), the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (R01 DK015556, to JAK), the Red Cooperative Research Thematic Network on Cancer (RTICC) (12/0036/0002 to XRB and 12/0036/0008 to XRB and MAP) and the Spanish Ministry of Health, Fund for Health Research–Institute of Health Carlos III (11/00951 to AU and 12/01528 to MAP).

Type 2 diabetes (T2D) mellitus and Alzheimer's disease (AD) are two prevalent diseases with comparable pathophysiological features and genetic predisposition. Patients with AD are more susceptible to develop T2D. However, the molecular mechanism linking AD and T2D remains elusive. In this study, we have generated a new mouse model to test the hypothesis that AD would prompt the onset of T2D in mice. To test our hypothesis, we crossed Alzheimer APPswe/PS1dE9 (APP/PS1) transgenic mice with mice partially deficient in leptin signaling (db/+). Body weight, plasma glucose, and insulin levels were monitored. Phenotypic characterization of glucose metabolism was performed using glucose and insulin tolerance tests. β-Cell mass, islet volume, and islet number were analyzed by histomorphometry. APP/PS1 coexpression in mice with intact leptin receptor signaling did not show any metabolic perturbations in glucose metabolism or insulin sensitivity. In contrast, APP/PS1 coexpression in db/+ mice resulted in nonfasting hyperglycemia, hyperinsulinemia, and hypercholesterolemia without changes in body weight. Conversely, fasting blood glucose and cholesterol levels remained unchanged. Coinciding with altered glucose metabolism, APP/PS1 coexpression in db/+ mice resulted in glucose intolerance, insulin resistance, and impaired insulin signaling. In addition, histomorphometric analysis of pancreata revealed augmented β-cell mass. Taken together, these findings provide experimental evidence to support the notion that aberrant Aβ production might be a mechanistic link underlying the pathology of insulin resistance and T2D in AD., This work was supported by grants from Instituto de Salud Carlos III (ISCIII)-Subdirección General de Evaluación y Fomento de la Investigación, Spain (PS09/00671 and CP08/00094), and a Europe-FP7 Marie Curie grant (IRG-247835) to I. Cózar-Castellano and by ISCIII-Subdirección General de Evaluación y Fomento de la Investigación, Spain (PS09/00969), RYC-2008- 02333, Proyecto de Excelencia Junta de Andalucía (P11-CTS-7847), and Fundación Dr. Eugenio Rodríguez Pascual to M. García-Alloza.

G protein-coupled receptor kinase 2 (GRK2) is emerging as a key integrative node in cell migration control. In addition to its canonical role in the desensitization of G protein-coupled receptors involved in chemotaxis, novel recently identified GRK2 substrates and interacting partners appear to mediate the GRK2-dependent modulation of diverse molecular processes involved in motility, such as gradient sensing, cell polarity or cytoskeletal reorganization. We have recently identified an interaction between GRK2 and histone deacetylase 6 (HDAC6), a major cytoplasmic a -tubulin deacetylase involved in cell motility and adhesion. GRK2 dynamically associates with and phosphorylates HDAC6 to stimulate its a -tubulin deacetylase activity at specific cellular localizations such as the leading edge of migrating cells, thus promoting local tubulin deacetylation and enhanced motility. This GRK2-HDAC6 functional interaction may have important implications in pathological contexts related to aberrant epithelial cell migration., Ministerio de Educación y Ciencia (SAF2011-23800); Fundación Ramón Areces; The Cardiovascular Network (RECAVA) of Ministerio Sanidad y Consumo-Instituto Carlos III (RD06-0014/0037); Comunidad de Madrid Indisnet Network (S2011/BMD-2332); Fundación Eugenio Rodriguez Pascual,; Fundación Areces; Instituto de Salud Carlos III (PI11/ 00859

Glioblastoma multiforme (GBM) is one of the deadliest forms of cancer, with an average survival time of approximately 1 year despite aggressive surgery, radiotherapy and chemotherapy. Here, we report a preclinical study by which the two main energy pathways of the tumor cells, oxidative phosphorylation and aerobic glycolysis, are simultaneously disrupted. The therapy is based on a plant gene encoding a β-glucosidase, linamarase (lis), which react with the substrate linamarin (lin) producing cyanide. We also use glucose oxidase (GO) to enhance oxidative stress and to induce cell death in the tumor. To test in vivo this suicide gene therapy system (lis/lin/GO), we used an orthotopic model of the human U87MG glioma cells, genetically modified to express the lis gene, and stereotactically implanted into the brains of nude rats (rnu/rnu). Despite its genetic condition, 6% of the animals immunorejected the xenotransplanted cells giving false curative results. We tried several delivery methods with limited success. The therapeutic cocktail, at dosages that perhaps eliminated the brain tumors, is too toxic for the animal causing its premature death., This work was supported by funds from: FINA Biotech; Fondo de Investigación Sanitaria, Ministerio de Sanidad y Consumo (PI06/0554) Spain; Ministerio de Ciencia e Innovación (SAF 2009-07259) Spain, and Fundación Eugenio Rodriguez Pascual (Madrid). The Centro de Biología Molecular S.O. is also the recipient of an institutional grant from the Ramón Areces Foundation. A.C. was the recipient of a grant MAEC-AECID, Spain.

This is an open-access article distributed under the terms of the Creative Commons Attribution License., [Background]: Three functional c-ras genes, known as c-H-ras, c-K-ras, and c-N-ras, have been largely studied in mammalian cells with important insights into normal and tumorigenic cellular signal transduction events. Two K-Ras mRNAs are obtained from the same pre-mRNA by alternative splicing. H-Ras pre-mRNA can also be alternatively spliced in the IDX and 4A terminal exons, yielding the p19 and p21 proteins, respectively. However, despite the Ras gene family’s established role in tumorigenic cellular signal transduction events, little is known about p19 function. Previous results showed that p19 did not interact with two known p21 effectors, Raf1 and Rin1, but was shown to interact with RACK1, a scaffolding protein that promotes multi-protein complexes in different signaling pathways (Cancer Res 2003, 63 p5178). This observation suggests that p19 and p21 play differential and complementary roles in the cell., [Principal Findings]: We found that p19 regulates telomerase activity through its interaction with p73a/b proteins. We also found that p19 overexpression induces G1/S phase delay; an observation that correlates with hypophosphorylation of both Akt and p70SK6. Similarly, we also observed that FOXO1 is upregulated when p19 is overexpressed. The three observations of (1) hypophosphorylation of Akt, (2) G1/S phase delay and (3) upregulation of FOXO1 lead us to conclude that p19 induces G1/S phase delay, thereby maintaining cells in a reversible quiescence state and preventing entry into apoptosis. We then assessed the effect of p19 RNAi on HeLa cell growth and found that p19 RNAi increases cell growth, thereby having the opposite effect of arrest of the G1/S phase or producing a cellular quiescence state., [Significance]: Interestingly, p19 induces FOXO1 that in combination with the G1/S phase delay and hypophosphorylation of both Akt and p70SK6 leads to maintenance of a reversible cellular quiescence state, thereby preventing entry into apoptosis., This work was supported by Fundacion de Investigacion Medica Mutua Madrileña Automovilista (Fundacion MMA), the Plan Nacional (MEC) BFU2005-00701 and the Fundacion Eugenio Rodriguez Pascual. M.C. was a recipient of a Fmed MMA fellowship.

OBJECTIVE: To compare the diet of Spanish children against the nutrient and food intake guidelines. To calculate an index of overall diet quality and check its validity against nutrient intake. DESIGN AND SETTING: Cross-sectional study in four cities in Spain, where information on food and nutrient intake was obtained from schoolchildren through a food frequency questionnaire. PARTICIPANTS: The sample included 1112 children (overall response rate of 85%) attending public and private schools and aged 6-7 y. Children were selected through random cluster sampling in schools, and stratified by sex and socioeconomic level. MAIN OUTCOME MEASURES: Mean nutrient intake, number of food servings, and the percentage of children who meet recommended nutrient and food-serving intake levels. The overall dietary quality was assessed using the Healthy Eating Index (HEI). RESULTS: Mean micronutrient intake exceeded 100% of the recommended dietary allowances, except for vitamin B6, which registered a mean intake of 77.1%. For almost all children, intake of saturated fat was above, and that of carbohydrate below, the recommended level, in contrast to the relatively high compliance with the recommendations for poly- and monounsaturated fatty acid, salt and fiber intake (69.7, 43.7, 40.7, and 30.1%, respectively). Consumption of food servings for each of the five American pyramid food groups came close to or exceeded USDA guidelines, with the exception of cereals, with 5.4 servings per day. The mean score obtained in the HEI was 64.6. Children who complied with all the food guide pyramid recommendations registered a higher dietary variety and a healthier nutritional profile. CONCLUSIONS: Children aged 6-7 y show scant compliance with the macronutrient goals for healthy eating. Micronutrient intake is adequate in general, yet there are small groups of children with risk of deficient intake of vitamins B6 and D. While Spanish children's eating habits are reasonably in line with American food guide pyramid guidelines, consumptions of cereals and fruit should be improved. This study was partly funded by grants from the Inter-national Olive Oil Board (Consejo Oleı ́cola Internacional),Comunidad de Madrid, Fundacio ́n Pedro Barrie ́de laMaza, and Fundacio ́n Eugenio Rodrı ́guez Pascual

The human malic enzyme (hME) promoter contains an inverted palindromic (IP4) 3,5,3′-triiodo-thyronine (T3) response element (T3RE) 15 bp downstream from an activating protein-1 (AP-1) site. The purpose of this study was to analyze the functional relationship between both cis-acting elements. The following observations indicate that these two elements operate as a functional unit in controlling the human ME gene: 1. Mutations within either the AP-1 or the T3RE sites inhibited basal as well as stimulated promoter activity. 2. Unliganded T3 receptors (TRβ) bind to the T3RE preferentially as homodimers and repress basal promoter activity. 3. The stimulation of the AP-1 driven promoter activity by TPA was perturbed by overexpression of TRβ. T3 failed to stimulate transcription above the basal levels in cells overexpressing either TRβ or TRβ/retinoid acid receptor (RXR), indicating that TRβ acts primarily as a transcriptional repressor in the context of the hME. Moreover, the finding of a repressive effect of TRβ without DNA binding suggests the existence of both DNA-dependent and independent mechanisms of TRβ-induced repression of transcription., This work has been supported in part by grants from the Dirección General de Investigación Científica y Técnica (DGICYT), and Fondo de Investigaciones Sanitarias (96/2014). C.G.-M. was a recipient of a research award from the the Fundación Rodríguez Pascual

[Background] P19 H-Ras, a second product derived from the H-Ras gene by alternative splicing, induces a G1/S phase delay, thereby maintaining cells in a reversible quiescence state. When P21 H-Ras is mutated in tumour cells, the alternative protein P19 H-Ras is also mutated. The H-Ras mutation Q61L is frequently detected in different tumours, which acts as constitutive activator of Ras functions and is considered to be a strong activating mutant. Additionally, a rare congenital disorder named Costello Syndrome, is described as a H-Ras disorder in children, mainly due to mutation G12S in p19 and p21 H-Ras proteins, which is present in 90 % of the Costello Syndrome patients. Our aim is to better understand the role of p19 and p21 H-Ras proteins in the cancer and Costello Syndrome development, concerning the miRNAs expression., [Methods] Total miRNAs expression regulated by H-Ras proteins were first analyzed in human miRNA microarrays assays. Previously selected miRNAs, were further analyzed in developed cell lines containing H-Ras protein mutants, that included the G12S Costello Syndrome mutant, with PCR Real-Time Taq Man miRNA Assays primers., [Results] This study describes how p19 affects the RNA world and shows that: i) miR-342, miR-206, miR-330, miR-138 and miR-99b are upregulated by p19 but not by p19W164A mutant; ii) anti-miR-206 can restore the G2 phase in the presence of p19; iii) p19 and p21Q61L regulate their own alternative splicing; iv) miR-206 and miR-138 are differentially regulated by p19 and p21 H-Ras and v) P19G12S Costello mutants show a clear upregulation of miR-374, miR-126, miR-342, miR-330, miR-335 and let-7., [Conclusions] These results allow us to conclude that the H-Ras G12S mutation plays an important role in miRNA expression and open up a new line of study to understand the consequences of this mutation on Costello syndrome. Furthermore, they suggest that oncogenes may have a sufficiently important impact on miRNA expression to promote the development of numerous cancers., This work was supported by the Plan Nacional (MEC) BFU2005-00701, FIS PI080007 and the Fundación Eugenio Rodríguez Pascual. M.C. was funded by an Fmed MMA fellowship. We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)